Construct design and ES cell targeting
miRNA genes have a great diversity in their genomic organization. About one-half of mammalian miRNAs are expressed from introns of annotated protein-coding genes (intronic miRNAs), whereas the others are found outside the context of an annotated gene (intergenic miRNAs). Intronic miRNAs can be transcribed via the promoter of their host gene, or be transcribed from an intronic promoter; intergenic miRNAs have their own promoter. Regardless of their genomic location, miRNAs are frequently found as polycistronic clusters.
There are at least 450 well-conserved miRNAs between mouse and human residing at more than 166 alleles. In this pipeline we focused on the mouse:human conserved subset of microRNAs, conditionally deleting polycistronic miRNA loci when possible. Given the relatively large numbers of miRKO [microRNA knockout] vectors required for these types of projects, we embraced high-throughput recombineering protocols. For most of the vectors we utilized the knockout-first approach derived by Testa and colleagues (Testa et al., 2004
), adapted to permit parallel preparation of many vectors in microtiter dish format (Fu et al., 2010
; Nefedov et al., 2011
). In this approach, sequential steps of genetic engineering do not require colony isolation and intermediate construct verification. We have generated 162 constructs targeting 194 miRNAs. This includes a second smaller subset of vectors targeting single miRNAs within a cluster (~30 miRNAs). So far we have generated conditional lacZ-reporter targeting vectors covering nearly 50% of the conserved miRNA genes.
Cre-lox technology was used to generate conditional miRNA knockout mice to ensure that gene function could be studied in a tissue- and temporal-specific manner. LoxP sites approximately 200–250 bases 5′ and 3′ proximal to the miRNA precursor (, middle panel) were placed in the constructs to achieve efficient conditional deletion with a Cre recombinase enzyme. For polycistronic miRNAs, loxP sites were placed outside of the first and the last miRNA precursor in the cluster. The length of polycistronic miRNAs ranged from a few hundred to hundreds of thousands of bases. To maximize efficient Cre recombination, miRNAs were targeted for individual deletion if their resident cluster was comprised of more than 2000 bases. A short 5′-arm (~1–2 kb) to aid PCR genotyping and a long 3′-arm (~10–15 kb) to boost specific targeting of the allele were chosen for homologous recombination.
ES cell targeting and PCR genotyping
A key addition to the vector design was the inclusion of a promoter-less lacZ reporter and a neomycin selection marker between the 5′ arm and the floxed miRNA precursor sequence, which could be removed with a Flp recombinase (, middle panel). This insertion allowed for the selection of the integrated transgene, and importantly provided the lacZ readout for transcription of the miRNA.
The majority of miRNA expression data have been generated by quantitative RT-PCR (qPCR), microarrays, and sequencing analyses using pools of isolated cells or tissue biopsy. In mammals, in vivo
expression data have been limited to the study of a small number of miRNAs, which were mainly studied during early embryo development by in situ
hybridization or the use of in vivo
miRNA ‘sensors’ (Mansfield et al., 2004
; Wienholds et al., 2005
). Due to the small size of miRNAs (22–23 nucleotides), the signal to background ratio of miRNA in situ
hybridization is very narrow and, therefore, it is difficult to achieve strong and specific signals in whole mount tissues. For this reason, we opted to design a promoter-less lacZ reporter such that it would indicate the activity of the endogenous miRNA promoter. This allowed the temporal and spatial detection of primary miRNA transcriptional
activity in mouse tissues or live cells. However, it is important to note that, similar to protein-encoding genes, transcriptional readout will not always be a true readout for the expression of the given gene, since miRNA expression may be regulated post-transcriptionally (Siomi and Siomi, 2010
Thus far 194 microRNAs (corresponding to 162 constructs) have been electroporated into embryonic stem cells. PCR screening is used to identify the properly targeted ES clones (PCR strategy shown in ). For the 5′ short arm, a three-primer PCR protocol was optimized to distinguish between non-targeted and targeted alleles. Given the technical challenge of long-range PCR across the entire ~10–15 kb long arm, the sequence integrity of the 3′-loxP site was verified as a proxy for 3′-end homologous recombination. PCR products from the targeted alleles were sequenced to validate the correct targeting. A diphtheria toxin gene was used as a negative selection marker, and Southern blot analyses were performed to ensure that there were no additional integrations at non-targeted loci (data not shown).
A 129/Ola-derived E14 ES cell line was used for targeting because of its history of highly efficient and reliable germ line transmission (Brenner et al., 2010
). We screened 46 ES colonies per construct as a first-pass. This first-pass allowed us to identify loci that were easily targeted and yielded at least 1–2 ES cell lines. This constituted approximately 20% of the targeted loci; those that did not pass the first screen were retargeted until the correctly targeted line(s) were identified.
Generation of knockout mice
To produce reporter-tagged knockouts and conditional knockouts from the same allele, targeting constructs were engineered to contain a combination of FRT and loxP sites (). By crossing a germline-transmitted lacZ-neo-flox mouse with a germline Flp deleter, the reporter and the selection marker can be removed to restore a functional wild type allele (this constitutes a conditional allele, where two loxP sites flank the intact miRNA). Performing this step is critical for intronic miRNAs, as the presence of the lacZ-neo cassette could adversely affect host gene expression, potentially complicating the interpretation of the phenotype. Conditional mice can be crossed either with special Cre strains to generate temporal or tissue-specific knockouts or with a germline Cre-deleter strain to generate constitutive knockouts. Crossing a germline-transmitted lacZ-neo-flox mouse to a Cre deleter line (lacZ-knockout allele) resulted in a knockout line that also reported on the transcription of the miRNA locus. This may be desirable in studies where miRNA expression patterns or the fate of cells harboring a deleted miRNA need to be tracked.
To verify the functionality of the recombination sites in vivo, we performed mating with either Flp or Cre germline deleter mice (). While Actin-Cre mice were very efficient in transgene excision (), Rosa-Flp mice showed a reduced efficiency (). Actin-Flp also displayed a similar level of recombination activity (data not shown). Incomplete penetrance by Flp recombinases may have been caused by the fact that the distance between FRT sites (~7 kb) is greater than between loxP sites (~3 kb). In any case, we were able to obtain the designed recombination by evaluating a litter or two.
To assess whether miRNAs are essential for normal development in general, we generated knockout-first mouse lines for 11 intergenic miRNA lines (). Among 11 lacZ-KO lines for intergenic miRNAs tested, we observed embryonic lethality only in the miR-205 lacZ-KO line. To ensure that the surviving homozygous lacZ-KO mice were true knockouts lacking specific miRNAs, we performed qPCRs using lacZ-positive tissues (). We observed near-complete ablations with the exceptions of the miR-30b/30d cluster and miR-7a-2. The remaining signals for miR-30b/30d and miR-7a-2 were most likely generated from related family members, since Southern analysis showed the insertion of the lacZ cassette into specific loci (data not shown). In fact, the miR-30 family is represented by six members (miR-30a, miR-30b, miR-30c-1, miR-30c-2, miR-30d, and miR-30e) and the miR-7 family has three members (miR-7a-1, miR-7a-2 and miR-7b). To confirm the ablation of the targeted miRNAs from related family members, investigators must employ qPCR measuring pre-miRNAs or deep sequencing approaches in the future analysis of miRNA knockouts.
Analysis of embryonic lethality due to loss of miRNAs.
The observed 9% lethality in our screen (1/11) is within the lowest range of estimates of essential genes reported for mice (8~20% based on ENU-mutagenesis screens(Alvarez-Saavedra and Horvitz, 2010
). Although the sample size is small, these data may suggest that deletion of most individual murine miRNAs do not lead to embryonic lethality or gross developmental defects. Similar findings have been reported for C. elegans (Miska et al., 2007
Another important consideration relates to the allele context. For example, miR-301a exists as an intronic miRNA, thus the generation of lacZ-KO mice by Cre recombinase would be expected to introduce an intronic stop cassette, likely altering host gene (fam33a
) expression. Since fam33a
is a known protein-coding gene with potential roles in cell cycle and cell division processes (Hanisch et al., 2006
), it is not possible to distinguish which gene(s) are responsible for embryonic lethality in this case. Thus, for ablation of intronic miRNAs, the appropriate experimental cross will be to employ actin-Flp recombination, followed by Cre-mediated knockout of the miRNA ().
Expression analysis in embryos
As an initial step towards understanding the in vivo function of miRNA genes, we performed expression analyses of the reporter in mouse embryos. The lacZ reporter can provide informative single cell expression patterns in complex tissue types such as the nervous and the immune system. To eliminate the interference of the neomycin cassette with miRNA transcription, we excised the neomycin cassette by crossing lacZ-neo-flox mice with actin-Cre mice (). The resulting lacZ-KO/+ males were crossed with wild type females, and the pregnant females were examined at two time points, 11.5 and 18.5 days post-coitum (E11.5 and E18.5). Embryos at E11.5 were chosen to study early developmental expression patterns, different germ layers, and overall body plan. Embryos at E18.5 were used to collect data on lacZ expression in major organs, including brain, sensory organs in the head, thymus, lung, heart, stomach, spleen, pancreas, intestine, liver, kidney, and bladder.
One third (6/18) of embryos displayed ubiquitous expression (for miR-30b/30d, miR-30e, miR-130a, miR-296/298, miR-301a, and miR-339), while approximately two thirds of the embryos showed distinct expression patterns. For miR-210 and miR-146a, we could not detect the lacZ reporter at either E11.5 or E18.5 stages ( and data not shown). Expression of miR-210 has been reported in some cancers and hypoxic conditions (Camps et al., 2008
; Giannakakis et al., 2008
) and miR-146a is mainly observed in a subset of immune and cancer cells (Jazdzewski et al., 2009
; Lu et al., 2010
; Taganov et al., 2006
), suggesting that they may not be expressed during normal development or they are expressed only in a subset of specialized tissues. In fact, the adult mice have shown lacZ activities in a subset of specialized immune cells (Figure S1
and unpublished data).
Expression analysis of miRNA lacZ reporter
We also conducted a limited but focused expression analysis in adult mice, investigating miRNA expression in defined subsets of hematopoietic cells isolated from tail blood using flow cytometry (Figure S1
). This single cell-based reporter assay was able to distinguish lacZ activities for the closely related family members such as miR-30 family. The lacZ expression detected in peripheral blood lymphocytes correlated with published expression data in 22 of the 25 mouse strains tested. It is important to note that we unexpectedly did not detect significant lacZ activity for miR-150 in lymphocytes. We suspect that the flow cytometry assay may not be sufficiently robust to detect low levels of lacZ expression, or perhaps the lacZ reporter cassette is silent at this locus.
The expression of miRNAs varies dynamically in the brain both before and after birth in mice (Krichevsky et al., 2003
; Miska et al., 2004
). As expected, we found several miRNAs that were exclusively expressed in the central nervous system (miR-7a-2, miR-135b, miR-325, and miR-688). In addition to the central nervous system, we observed interesting reporter expression patterns in the respiratory system (miR-141/200c and miR-497/195). We performed limited histological analysis for a handful of lacZ-stained tissues either using vibratome sectioning or traditional paraffin-sectioning and confirmed that the lacZ reporter expression is very specific to a subset of cells in a given tissue and easily tractable with a single cell resolution (data not shown). Detailed expression profiling of tissue-specific miRNAs may shed light on the roles of miRNA genes in that sub-compartment. In addition, the reporter expression pattern will guide the selection of Cre lines for conditional knockouts.
In addition to differential spatial expression patterns, temporal regulation of miRNA expression was observed for several miRNAs in a variety of tissues (miR-688, miR-654/376b, and miR-130b/301b). A more detailed analysis of temporal expression changes may provide additional clues about their function.